Currently we lack biomarkers to predict development of Estrogen Receptor-negative [ER(-)] invasive breast cancer. Progress in this area is hindered by our lack of understanding of the biology of ER(-) breast cancer initiation/progression. This application studies the regulation of ER(-) breast cancer invasion by a novel signaling network involving Snail-1, a transcriptional repressor silenced by ER. In Aim 1, we will investigate the regulation of ER(-) breast tumor cell invasion by a novel signaling network involving two transcriptional repressors (Snail-1 and ZEB1). Snail-1 and ZEB1 promote invasive tumor behavior by suppressing transcription of epithelial genes. Snail-1 and ZEB1 are frequently co-expressed in breast cancer cell lines, but it is unknown how they cooperate to promote invasive behavior. MicroRNA200 (miRNA200) family members suppress breast tumor cell invasion, and their expression levels are reduced in invasive relative to non-invasive breast tumor cells. However, the factors that initially suppress miRNA200 in breast tumor cells, thus driving ZEB1 expression and invasive behavior, are unknown. We will investigate the hypothesis that Snail-1 promotes ER(-) breast tumor cell invasion by regulating the miRNA200/ZEB1 axis. To validate our in vitro findings, we will investigate expression of the nuclear Snail-1/miRNA200/nuclear ZEB1 network in primary human ER(-) breast cancers, and retrospectively determine if expression of this network predicts patient prognosis. Snail-1 mRNA levels do not reflect Snail-1 activity because: 1) Snail-1 activity is dependent on its nuclear localization, and 2) Snail-1 localization is regulated by Snail-1 post-translational modifications. Previous studies of Snail-1 protein expression in human cancers used poorly-characterized antibodies that in some cases react with other Snail-1 family members. We have optimized an immunohistochemistry protocol for detecting nuclear Snail-1 protein using a highly-specific Snail-1 antibody. Our preliminary studies indicate that nuclear Snail-1 is expressed frequently in ER(-) but not in ER(+) ductal breast cancers. In Aim 2, we will study expression of this network in pre-malignant breast lesions obtained from a unique cohort of women at high risk for developing breast cancer. We will test if expression of this network in these pre-malignant lesions retrospectively predicts subsequent development of invasive ER(-) breast cancer. The results from this two-year study will allow us to perform power calculations necessary for a future prospective study investigating the value of a nuclear Snail-1/miRNA200/nuclear ZEB1 network as a biomarker in pre-malignant lesions that predicts development of invasive ER(-) breast cancer. Collectively, the studies in this two-year proposal will define molecular determinants of ER(-) breast tumor cell invasive behavior as a means of identifying: 1) biomarkers for early detection of invasive breast cancer, and 2) targets for prevention and treatment of invasive ER(-) breast disease. PUBLIC HEALTH RELEVANCE: Only a fraction of women diagnosed with pre-malignant breast lesions develop invasive breast tumors. Currently we lack biomarkers that predict a high risk for invasive tumor growth. Identification and validation of such a biomarker will require: 1) molecular studies of key regulators of invasion, 2) studies determining the frequency of expression of these invasion markers in pre-malignant and malignant lesions, and 3) prospective studies determining if expression of these markers in pre-malignant lesions predicts subsequent development of invasive cancer. The experiments in this two year proposal will: 1) investigate the regulation of Estrogen Receptor-negative [ER(-)] breast cancer invasion by a novel signaling network involving the transcriptional repressor Snail-1, 2) examine the frequency of expression of determinants of this Snail-1 network in malignant and pre-malignant breast lesions, and 3) retrospectively examine if expression of this Snail-1 network in pre-malignant breast lesions predicts development of invasive ER(-) breast cancer. Using the results obtained, we will perform a power calculation necessary for planning a future prospective study that tests if this signaling network is a valuable biomarker for predicting development of invasive ER(-) breast cancer. Collectively, these studies will define molecular determinants of ER(-) breast tumor cell invasive behavior as a means of identifying: 1) biomarkers for early detection of invasive ER(-) breast cancer, and 2) targets for prevention and treatment of invasive ER(-) breast disease.